Expression, purification and crystallization of a protein resulting from the inversion of the amino‐acid sequence of a helical bundle

• Published in: Acta crystallographica. Section F, Structural biology communications Vol. 73; no. 1; pp. 51 - 53
• Main Authors: Kefala, Aikaterini, Kotsifaki, Dina, Providaki, Mary, Amprazi, Maria, Kokkinidis, Michael
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England International Union of Crystallography 01.01.2017; Wiley Subscription Services, Inc
• Subjects: Amino Acid Sequence; Amino acids; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bundling; Cloning, Molecular; Crystallization; Crystallography; Crystallography, X-Ray; Crystals; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Helices; heptad repeat; hyperthermophilic protein; Inverse; Inversions; Plasmids - chemistry; Plasmids - metabolism; Protein Conformation, alpha-Helical; Protein Engineering; protein folding; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Research Communications; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Rop protein; Sequence Inversion; sequence–structure relationship; X-Ray Diffraction; sequence–structure relationship; Rop protein; heptad repeat; protein folding; hyperthermophilic protein
• ISSN: 2053-230X; 2053-230X
• DOI: 10.1107/S2053230X16020173

Earlier studies have found that the occurrence of inverse sequence identity in proteins is not indicative of three‐dimensional similarity, but rather leads to different folds or unfolded proteins. Short helices, however, frequently keep their conformations when their sequences are inverted. To explore the impact of sequence inversion on long helices, revRM6, with the inverse amino‐acid sequence relative to RM6, a highly stable variant of the ColE1 Rop protein, was engineered. RM6 is a highly regular four‐α‐helical bundle that serves as a model system for protein‐folding studies. Here, the crystallization and preliminary crystallographic characterization of revRM6 are reported. The protein was overexpressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belonged to space group P41212, with unit‐cell parameters a = b = 44.98, c = 159.74 Å, and diffracted to a resolution of 3.45 Å. To explore the sequence–structure relationships of four‐α‐helical bundles, the revRM6 protein, which has been engineered with the exact inverse amino‐acid sequence of a hyperthermophilic helical bundle, was expressed, purified and crystallized and X‐ray diffraction data were collected.